Mounting evidence suggests that the tumor environment can inhibit the development of an effective immune response by promoting T cell dysfunction. Studies from our laboratory and several others suggest that the ability of tumors and their products to promote T cell death represents an important mechanism of immune dysfunction. Flow cytometry analysis of tumor infiltrating T cells stained with AnnexinV/7AAD as well as in situ TUNEL staining revealed that a high percentage of these cells showed signs of apoptosis/necrosis, suggesting that the tumor microenvironment has a deleterious effect on tumor infiltrating T cells (TILs), although the immunosuppressive effects of RCC extend into the periphery as well. Additional studies demonstrated that in patients with active disease the T cell response to MAGE-6 and EphA2, expressed by renal cell carcinomas was predominately Type-2 (IL-4, IL-5.IL-10) rather than a type-1(IFNg) which is known to be critical for the development of effective anti-tumor immunity. However, patients rendered disease free by surgery displayed Th-1 responses (IFNg, ELISPOT). HLA-DR4/tumor peptide tetramer studies showed that the MAGE-6 and EphA2 specific CD4+T cells were highly sensitive to apoptosis compared to T cells binding influenza peptides and the bulk peripheral blood T cell population. Our data also suggest that gangliosides play in important role in tumor-induced immune dysfunction. Gangliosides constitute over 50% of the apoptotic activity induced by supernatants from RCC explants and by RCC lines. RCC derived gangliosides can also induce a Th2 bias in vitro. HPLC with on line mass spectrometry analysis suggests that there are several gangliosides, including GM2, which mediate the immunosuppression. Here we plan to determine which of the common gangliosides derived from RCC tissue, supernatant and plasma are the most effective at inducing T cell death, a Type-2 bias, and whether the bias is related to the apoptosis of the MAGE-6 and EphA2 specific Th-1 T cells. We will also determine whether the immune suppression is mediated by select gangliosides shedding from tumor and associating with lipid rafts on the plasma membranes of T cells and whether expression of the inhibitory gangliosides correlates with the Th-2 bias and increased sensitivity of patient T cells to apoptosis (aim1). Whether T cell death induced by ganglosides results from degradation to ceramide or to their migration to mitochondria will be examined (aim2). The observation that the anti-oxidant deferoxamine (DFO) can block ganglioside-mediated apoptosis of T cells will be studied further to define how DFO protects T cells from apoptosis. We will also define the sequence of events leading to T cell death, including the role of iron and lysosomes in this process (aim 3)